Method for predicting response to epidermal growth factor receptor-directed therapy

ABSTRACT

This invention provides methods for determining or predicting response to cancer therapy in an individual.

This application is a divisional application of U.S. Divisionalapplication Ser. No. 11/548,386, Oct. 11, 2006, which claims the benefitof U.S. application Ser. No. 10/600,129, Jun. 19, 2003, which claims thebenefit of U.S. Provisional Application Ser. No. 60/389,796, filed Jun.19, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for predicting the response to cancertherapy in an individual.

2. Background of the Invention

Cellular growth and differentiation processes involve growth factorsthat exert their actions through specific receptors expressed in thesurfaces of responsive cells. Ligands binding to surface receptors, suchas those that carry an intrinsic tyrosine kinase activity, trigger acascade of events that eventually lead to cellular proliferation anddifferentiation (Carpenter et al., Biochem., 48: 193-216, 1979; Sachs etal., Cancer Res., 47: 1981-1986, 1987). Receptor tyrosine kinases can beclassified into several groups on the basis of sequence similarity anddistinct features. One of these groups includes the epidermal growthfactor receptor family, which included erbB-1 (EGFR or HER-1) (Carpenteret al., Biochem., 48: 193-216, 1979); erbB-2 (HER-2/neu) (Semba et al.,Proc. Natl. Acad. Sci., 82: 6497-6501, 1985; Coussens et al., Science,230: 1130-1139, 1985, Bargmann et al., Cell, Vol. 45, 649-657, 1986);erbB-3 (HER-3) (Kraus et al., Proc. Natl. Acad. Sci., 86: 9193-9197,1989; Carraway et al., R. A. J. Biol. Chem., 269: 14303-14306, 1994),and erbB-4 (HER-4) (Plowman et al., Nature, 366: 473-475, 1993; Tzaharet al., Biol. Chem., 269: 25226-25233, 1994).

As an example of a ligand that can bind to surface receptors, NDF (neudifferentiation factor)/Heregulin is a receptor tyrosine kinase ligandthat can stimulate the tyrosine phosphorylation of erbB-2 throughheterodimerization with its receptors erbB-3 or erbB-4 (Peles, et al.,Cell, 69:205-216, 1992, Peles, et al., EMBO J. Mar; 12(3):961-71. 1993;Holmes et al, Science, 256:1205-1210, 1992. Tzahar et al., Biol. Chem.,269: 25226-25233, 1994; Plowman et al., Nature, 366: 473-475, 1993;Pinkas-Kramarski et al., Proc. Natl. Acad. Sci., 91:9387-9391, 1994;Pinkas-Kramarski et al., The Journal of Biological Chemistry, Vol. 271,No. 32: 19029-19032, 1996; Pinkas-Kramarski et al., Oncogene, 16,1249-1258, 1998.). Depending on the cell line studied, NDF/Heregulin caneither elicit a growth arrest and differentiation phenotype, resultingin morphological changes, induction of lipids, and expression ofintracellular adhesion molecule-1, or induce a mitogenic response(Holmes et al., Science, 256:1205-1210, 1992; Peles et al., Cell,69:205-216, 1992; Bacus et al., Cancer Res. 53:5251-5261, 1993).

Activation of erbB receptor heterodimers is coupled to and stimulatesdownstream MAPK-Erk1/2 and PI3K-AKT growth and survival pathways whosederegulation in cancer has been linked to disease progression andrefractoriness to therapy (Olayioye, M. A., et al., Mol. Cell. Biol. 18,5042-5051 (1998), Fukazawa, T., et al., J. Biol. Chem. 271, 14554-14559(1996), Hackel, P. O., et al., Curr. Opin. Cell Biol. 11, 184-189(1999); Tzahar, E., et al., Mol. Cell. Biol. 16, 5276-5287 (1996);Lange, C. A., et al., J. Biol. Chem. 273, 31308-31316 (1998). Forexample, HER-3 is a major docking site for phoshoinositide-3-kinase(PI3K). In addition, NDF/Heregulin stimulation causes activation of thePI3K pathway and phosphorylation of AKT (Altiok et al., J. Biol. Chem.,274, 32274-32278, 1999; Liu et al., Res. Comm., 261, 897-903, 1999; Xinget al., Nature Med., 6, 189-195, 2000). These observations implicatePI3K/AKT in the signaling cascade that results from HER-3heterodimerization with overexpressed HER-2/neu receptors in breastcancer cells; activation of PI3K/AKT promote cell survival and enhancedtumor aggressiveness (Shak, Semin. Oncol., Suppl 12:71-77, 1999; Huanget al., Clinical Cancer Res., Vol. 7: 2166-2174, 2000). In addition,AKT2 was reported to be activated and overexpressed inHER-2/neu-overexpressing breast cancers (Bacus et al., Oncogene, 21:3532-3540, 2002).

Most tumors of epithelial origin express multiple erbB (HER) receptorsand co-express one or more EGF-related ligands suggesting that autocrinereceptor activation plays a role in tumor cell proliferation. Becausethese ligands activate different erbB/HER receptors, it is possible thatmultiple erbB receptor combinations might be active in a tumor, acharacteristic that could influence its response to an erbB-targetedtherapeutic. For example, erbB-2/HER-2 is overexpressed in 20 to 30% ofall breast cancers, and its overexpression is associated with poorprognosis, suggesting that it could be used as a target for anti-tumoragents (Slamon et al., Science, 235: 177-182, 1987; Tagliabue et al.,Int. J. Cancer, 47: 933-937, 1991; Hudziak et al., Mol. Cell. Biol., 9:1165-1172, 1989). Studies have shown that in erbB-2 overexpressingbreast cancer cells, treatment with antibodies specific to HER-2/erbB-2in combination with chemotherapeutic agents (e.g., cisplatin,doxoubicin, taxol) elicits a higher cytotoxic response than treatmentwith chemotherapy alone (Hancock et al., Cancer Res., 51: 4575-4580,1991; Arteaga et al., Cancer, 54:3758-3765, 1994; Pietras et al.,Oncogene, 9: 1829-1838, 1994). One possible mechanism by whichHER-2/erbB-2 antibodies might enhance cytotoxicity to chemotherapeuticagents is through the modulation of the HER-2/erbB-2 protein expression,(Bacus et al., Cell Growth & Diff., 3: 401-411, 1992, Bacus et al.,Cancer Res. 53:5251-5261, 1993; Stancovski et al., Proc Natl Acad SciUSA 88: 8691-8695, 1991; Klapper et al., Oncogene 14, 2099-2109, 1997,and Klapper et al., Cancer Res., 60: 3384-3388, 2000), or by interferingwith DNA repair (Arteaga et al., Cancer, 54:3758-3765, 1994, and Arteagaet al., J Clinical Oncology, Vol. 19, No 18 s, 32 s-40 s, 2001; Pietraset al., Oncogene, 9: 1829-1838, 1994).

Because of the effect of anti-HER-2/erbB-2 antibodies on cellulargrowth, a number of approaches have been used to therapeutically targetHER-2/erbB-2 or EGFR overexpressing cancers. For clinical use, oneapproach is to interfere with the kinase activity of the receptor byusing inhibitors that block the nucleotide binding site of HER-2/neu orEGFR (Bruns, et al., Cancer Research, 60, 2926-2935, (2000);Christensen, et al, Clinical Cancer Research, Vol. 7, 4230-4238, 2001,Erlichman, et al., Cancer Research 61, 739-748, 2001, Fujimura, et al.,Clinical Cancer Research, Vol. 8, 2448-2454, 2002; Herbst, et al.,Journal of Clincal Oncology, Vol. 20, No. 18, 3815-3825, 2002; Hidalgo,et al, J. Clinical Oncology, Vol 19, No 13: pp 3267-3279, 2001; Moasser,et al, Cancer Res., 61: 7184-7188, 2001; Normanno, et al, Ann. ofOncol., 13: 65-72, 2002). A second approach is using ansamycins toinfluence the stability of HER2/neu receptors (Munster, et al., CancerResearch 62, 3132-3137, 2002; Basso et al, Oncogene, 21: 1159-1166,2002). Another approach is the use of antibodies directed to variouserbB receptors specifically EGFR or HER-2/neu (Alaoui-Jamali, et alBiochem. Cell. Biol., 75:315-325, 1997; Albanell, et al., J. NationalCancer Institute, Vol 93, No. 24, 1830-31, 2001; Baselga, et al.,Pharmacol Ther 64: 127-154, 1994 and Baselga, et al., Annuals ofOncology 13: 8-9, 2002; Mendelsohn, Seminars in Cancer Biology, Vol. 1,pp. 339-344, 1990). A number of monoclonal antibodies and smallmolecule, tyrosine kinase inhibitors targeting EGFR or erbB-2 have beendeveloped. For example, HERCEPTIN® is approved for treating the 25% ofwomen whose breast cancers overexpress erbB-2 protein or demonstrateerbB-2 gene amplification (Cobleigh, M. A., et al., J. Clin. Oncol. 17,2639-2648 (1999)). Analysis of various antibodies to HER-2/neu has ledto the identification of the murine monoclonal, 4D5. This antibodyrecognizes an extracellular epitope (amino acids 529 to 627) in thecysteine-rich II domain that resides very close to the transmembraneregion. Treatment of breast cancer cells with 4D5 partially blocksNDF/heregulin activation of HER-2-HER-3 complexes, as measured byreceptor phosphorylation assays. To allow for chronic humanadministration, murine 4D5 was humanized to generate HERCEPTIN®(trastuzumab) (Sliwkowski et al, Sem. in Oncol., 26:60-70, 1999; Ye etal., Oncogene, 18: 731-738, 1999; Carter et al, Proc. Natl. Acad Sci USA89:4285-4289, 1992; Fujimoto-Ouchi et al, Cancer Chemother Pharmacol,49: 211-216, 2002; Vogel, et al., Oncology, 61(suppl 2):37-42, 2001;Vogel, et al., Journal of Clinical Oncology, Vol 20, No. 3:719-726,2002). In addition, several EGFR-targeted therapies are currently underclinical investigation (Mendelsohn, J., & Baselga, J., Oncogene 19,6550-6565 (2000); Xia, W., et al. Oncogene 21, 6255-6263 (2002)). Inparticular, a human anti-EGFr monoclonal antibody, designated ABX-EGF(and also referred to herein as ABX-0303, as described in detail in U.S.Pat. No. 6,235,883; the disclosure of which is hereby incorporated byreference), is being developed by Abgenix, Inc. and Immunex Corporation(Yang X et al. Development of ABX-EGF, a fully human anti-EGF receptormonoclonal antibody, for cancer therapy. Crit. Rev Oncol Hemato38(1):17-23 (2001); Yang X-D et al. Eradication of Established Tumors bya Fully Human Monoclonal Antibody to the Epidermal Growth FactorReceptor without Concomitant Chemotherapy. Cancer Research59(6):1236-1243 (1999)).

Historically, cytotoxic cancer therapies have been developed based onmaximum tolerated doses (MTD), treating patients without understandingthe tumor profile for likely responders. Hence, patients were oftensubjected to toxic therapies with limited therapeutic benefit. Recently,elucidating tumor growth and survival pathways has led to thedevelopment of tumor-targeted therapies. An example of this approach isGleevec™ an inhibitor of the c-abl family of tyrosine kinases approvedfor treating chronic myeloid leukemia and gastrointestinal stromaltumors (Druker, B. J. et al., N Engl. J. Med. 344, 1031-1037 (2001);Demitri, G. D., et al.; N. Engl. J. Med. 347, 472-480 (2002)).

In contrast, most erbB-receptor targeted therapies primarily exertcytostatic anti-tumor effects, necessitating their chronicadministration. Identification of biologically effective doses (BED),the dose or dose range that maximally inhibits the intended target,beyond which dose escalation is likely to add toxicity without benefit,is therefore essential. Moreover, many of these agents will be used incombination with cytotoxic therapies, where added toxicity may not betolerable, further supporting BED-based dosing. Targeted-therapy impliesthat populations of likely responders exists, and can be identified.

In view of the severe and deleterious consequences of administering aninappropriate or ineffective therapy to a human cancer patient, thereexists a need in the art for predicting the response to cancer therapyin an individual.

SUMMARY OF THE INVENTION

This invention provides methods for predicting a response of anindividual to a particular cancer treatment regimen.

In a first aspect, the invention provides methods for predicting aresponse to an epidermal growth factor receptor-directed therapy in ahuman subject, the method comprising the step of assaying a tumor samplefrom the human subject before therapy with one or a plurality ofreagents that detect expression and/or activation of predictivebiomarkers for cancer; and determining a pattern of expression and/oractivation of at least two of said predictive biomarkers, wherein thepattern predicts the human subject's response to the epidermal growthfactor receptor-directed therapy. In certain embodiments, the predictivebiomarker is a growth factor receptor, or a growth factorreceptor-related downstream signaling molecule. The growth factorreceptors can be HER1 (EGFR), pHER1, HER2/neu, HER3, or any combinationthereof. The growth factor receptor-related downstream signalingmolecules can be pERK. In further embodiments, the predictive biomarkersare HER1 (EGFR), pHER1, HER2/neu, HER3, or pERK, or any combinationthereof.

In further embodiments, the predictive biomarkers are HER1 (EGFR) andHER3. In other embodiments, when HER1 (EGFR) is undetectable, it ispredictive of the human subject not responding to the epidermal growthfactor receptor-directed therapy. In still other embodiments, whereinwhen HER3 is undetectable, it is predictive of the human subjectresponding to the epidermal growth factor receptor-directed therapy. Infurther embodiments, the predictive biomarkers are HER1 (EGFR) and pERK;or the predictive biomarkers are pERK and HER3, or the predictivebiomarkers are HER1 (EGFR), HER3, and pERK.

In a second aspect, the invention provides a kit for the determining aresponse to an epidermal growth factor receptor-directed therapy in asubject, wherein the kit comprises at least two reagents that detectexpression and/or activation of predictive biomarkers for cancer. Incertain embodiments, the kit comprises three reagents. In otherembodiments, the predictive biomarkers are HER1, HER3, or pERK, or anycombination thereof.

In a third aspect, the invention provides methods for predicting aresponse to a cancer therapy in a human subject, the method comprisingthe step of assaying a cell or tissue sample from the human subjectbefore therapy with one or a plurality of reagents that detectexpression and/or activation of predictive biomarkers for cancer,wherein said predicative biomarkers consist of growth factor receptorligands; and determining a pattern of expression and/or activation of atleast two of said predictive biomarkers, wherein the pattern predictsthe human subject's response to the cancer therapy. In otherembodiments, the growth factor receptors are HER1 (EGFR), pHER1,HER2/neu, HER3 or any combination thereof. In still other embodiments,the cancer therapy is an epidermal growth factor receptor-directedtherapy. In further embodiments, the cancer therapy is an anti-EGFRantibody. Further, the antibody is ABX-0303.

In a fourth aspect, the invention provides methods of selecting asubject with cancer for treatment with a molecule targeting epidermalgrowth factor receptor (EGFR), comprising determining the level ofexpression of HER3 in a cell or tissue sample from the subject, whereinif the level of HER3 expression is low in the cells, the subject isselected. In other embodiments, the molecule is an anti-EGFR antibody.Further, the antibody is ABX-0303. In still other embodiments, thedetermining step further comprises determining expression of one or moreof HER1 (EGFR), pHER1, HER2/neu, and pERK.

In a fifth aspect, the invention provides method of predicting thelikely response rate to a molecule targeting epidermal growth factorreceptor (EGFR) of a subject having a cancer that overexpresses EGFR,comprising the step of determining the level of expression of HER3 in acell or tissue sample from the subject, wherein if the level of HER3expression is low in the cells, the subject is likely to respond to themolecule targeting EGFR. In other embodiments, the molecule is ananti-EGFR antibody. Further, the antibody is ABX-0303. In still otherembodiments, the determining step further comprises determiningexpression of one or more of HER1 (EGFR), pHER1, HER2/neu, and pERK.

In a sixth aspect, the invention provides methods of treating a subjectwith cancer, comprising determining the level of expression of HER3 inthe cells from the subject, and treating the subject with an anti-EGFRantibody when HER3 expression levels in the cell are low. In furtherembodiments, the antibody is ABX-0303. In other embodiments, thedetermining step further comprises determining expression of one or moreof HER1 (EGFR), pHER1, HER2/neu, and pERK. Further, the antibody isABX-0303. In still other embodiments, the level of expression of HER3 isundetectable. Further, the antibody is ABX-0303.

In a seventh aspect, the invention provides methods of selecting asubject with cancer for treatment with a molecule targeting epidermalgrowth factor receptor (EGFR), the method comprising:

-   -   a) determining an expression and/or activation profile of two or        more growth factor receptors in cells and/or tissues of the        subject; and    -   b) selecting the subject based on the expression and/or        activation profile,        wherein the subject is selected when the level of expression of        HER3 is low, the level of expression of the HER1 is high, and/or        the level of the pERK index is high. In other embodiments, the        molecule is an anti-EGFR antibody. Further, the antibody is        ABX-0303. In another aspect, the growth factor receptors        comprise one or more of HER1 (EGFR), pHER1, HER2/neu, and HER3.

Specific embodiments of the present invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the response to ABX-0303 by a patient with elevatedHER1 and pERK, and decreased levels of HER3. The figure representsquantitative immunohistochemical analysis of EGFR, pEGFR, HER2, HER3,and pERK.

FIG. 2 illustrates the response to ABX-0303 by a patient with elevatedHER1, HER3, and pERK. The figure represents quantitativeimmunohistochemical analysis of EGFR, pEGFR, HER2, HER3, and pERK.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

This invention provides methods for predicting response in cancersubjects to cancer therapy, including human cancer patients.

In contrast to traditional anticancer methods, where chemotherapeuticdrug treatment is undertaken as an adjunct to and after surgicalintervention, neoadjuvant (or primary) chemotherapy consists ofadministering drugs as an initial treatment in cancer patients. Oneadvantage of such an approach is that, for primary tumors of more than 3cm, it permits the use of conservative surgical procedures (as opposedto, e.g., radical mastectomy in breast cancer patients) for the majorityof patients, due to the tumor-shrinking effect of the chemotherapy.Another advantage is that for many cancers, a partial and/or completeresponse is achieved in about two-thirds of all cases. Finally, becausethe majority of patients are responsive after two to three cycles ofchemotherapeutic treatment, it is possible to monitor the in vivoefficacy of the chemotherapeutic regimen employed, which is importantfor a timely identification of those cancers which are non-responsive tochemotherapeutic treatment. Timely identification of non-responsivetumors, in turn, allows the clinician to limit the cancer patient'sexposure to unnecessary side-effects of treatment and to institutealternative treatments. However, the methods present in the art,including histological examination, are insufficient for such a timelyand accurate identification. The present invention provides methods bywhich a more informed and effective regime of therapy can beadministered.

A cancer diagnosis, both an initial diagnosis of disease and subsequentmonitoring of the disease course (before, during, or after treatment) isconventionally confirmed through histological examination of cell ortissue samples removed from a patient. Clinical pathologists need to beable to accurately determine whether such samples are benign ormalignant and to classify the aggressiveness of tumor samples deemed tobe malignant, because these determinations often form the basis forselecting a suitable course of patient treatment. Similarly, thepathologist needs to be able to detect the extent to which a cancer hasgrown or gone into remission, particularly as a result of or consequentto treatment, most particularly treatment with chemotherapeutic orbiological agents.

Histological examination traditionally entails tissue-stainingprocedures that permit morphological features of a sample to be readilyobserved under a light microscope. A pathologist, after examining thestained sample, typically makes a qualitative determination of whetherthe tumor sample is malignant. It is difficult, however, to ascertain atumor's aggressiveness merely through histological examination of thesample, because a tumor's aggressiveness is often a result of thebiochemistry of the cells within the tumor, such as protein expressionor suppression and protein activation, which may or may not be reflectedby the morphology of the sample. Therefore, it is important to be ableto assess the biochemistry of the cells within a tumor sample. Further,it is desirable to observe and quantitate both gene expression andprotein activation of tumor related genes or proteins, or morespecifically cellular components of a tumor-related signally pathway.

Cancer therapy can be based on molecular profiling of tumors rather thanhistology or site of disease. Elucidating the biological effects oftargeted-therapies in tumor tissue and correlating these effects withclinical response helps identify the predominant growth and survivalpathways operative in tumors, thereby establishing a profile of likelyresponders and conversely providing a rational for designing strategiesto overcoming resistance.

It is necessary to consider additional biomarkers beyond the presence ofthe target, such as EGFR, for subjects who are considered for treatmentwith, for example, biomolecules that modulate EGFR. Not all tumor cellsrespond to inhibition of ErbB receptors, despite exhibiting aberrantErbB-1 and/or ErbB-2 expression. Examples include MKN7 and BT474 ErbBreceptor-overexpressing carcinoma cell lines, wherein BT474 cellsrespond to HERCEPTIN® but MKN7 cells do not. These observations haveclear implications for erbB-directed therapeutics and the considerationof the expression of multiple erbB receptors and in tumors.

For example, ABX-0303 (as referred to herein as ABX-EGF), an epidermalgrowth factor receptor-directed therapy sponsored by Abgenix and ImmunexCorporation, effectively targets HER1 to prevent the growth of renalcell cancers. Based on the positive correlation between pERK expressionand response to ABX-0303, it is likely that HER1 is acting through theMAPK pathway. In addition, HER3 was found to be elevated in a largepercentage of renal biopsies analyzed from non-responders. Onepossibility is that HER3 is interacting with HER2 to confound the actionof the drug.

Automated (computer-aided) image analysis systems known in the art canaugment visual examination of samples. In a representative system, thecell or tissue sample is exposed to detectably labeled reagents specificfor a particular biological marker, and the magnified image of the cellis then processed by a computer that receives the image from acharge-coupled device (CCD) or camera such as a television camera. Sucha system can be used, for example, to detect and measure expression andactivation levels of Herl, pHER1HER2, HER3, and pERK in a sample.Additional biomarkers are also contemplated by this invention. Thismethodology provides more accurate diagnosis of cancer and a bettercharacterization of gene expression in histologically identified cancercells, most particularly with regard to expression of tumor marker genesor genes known to be expressed in particular cancer types and subtypes(i.e., different degrees of malignancy). This information permits a moreinformed and effective regimen of therapy to be administered, becausedrugs with clinical efficacy for certain tumor types or subtypes can beadministered to patients whose cells are so identified.

For example, expression and activation of proteins expressed fromtumor-related genes can be detected and quantitated using methods of thepresent invention. Further, expression and activation of proteins thatare cellular components of a tumor-related signaling pathway can bedetected and quantitated using methods of the present invention.Further, proteins associated with cancer can be quantified by imageanalysis using a suitable primary antibody against biomarkers, such as,but not limited to, Her-1, Her-2, p-Her-1, Her-3, or p-ERK, and asecondary antibody (such as rabbit anti-mouse IgG when using mouseprimary antibodies) and/or a tertiary avidin (or Strepavidin) biotincomplex (“ABC”).

In practicing the method of the present invention, staining procedurescan be carried out by a technician in the laboratory. Alternatively, thestaining procedures can be carried out using automated systems. Ineither case, staining procedures for use according to the methods ofthis invention are performed according to standard techniques andprotocols well-established in the art.

By “cell or tissue sample” is meant biological samples comprising cells,most preferably tumor cells, that are isolated from body samples, suchas, but not limited to, smears, sputum, biopsies, secretions,cerebrospinal fluid, bile, blood, lymph fluid, urine and faeces, ortissue which has been removed from organs, such as breast, lung,intestine, skin, cervix, prostate, and stomach. For example, a tissuesample can comprise a region of functionally related cells or adjacentcells.

The amount of target protein can then be quantitated by the averageoptical density of the stained antigens. Also, the proportion orpercentage of total tissue area stained may be readily calculated, asthe area stained above an antibody threshold level in the second image.Following visualization of nuclei containing biomarkers, the percentageor amount of such cells in tissue derived from patients after treatmentmay be compared to the percentage or amount of such cells in untreatedtissue or said tissue prior to treatment. For purposes of the inventionherein, “determining” a pattern of expression and/or activation of abiomarker is understood broadly to mean merely obtaining the informationon such biomarker(s), either through direct examination or indirectlyfrom, for example, a contract diagnostic service.

Thus, the level of expression and/or activation in a cell can bedetermined by, for example, quantitative immunohistochemistry. In thiscase, the level of expression of HER1, HER2, and/or HER3 is consideredto be low if the OD is less than 9. Further, the level of expression isalso considered to be low if the OD is less than 5, or less than 3, orif the OD is 0 (undetectable). In addition, the level of expression ofHER1, HER2, and/or HER3 is considered to be high is the OD is greaterthan 9. Further, the level of expression can be considered high for pERKwhen the pERK index is greater than 99.

Particularly useful embodiments of the present invention and theadvantages thereof can be understood by referring to Examples 1-7. TheseExamples are illustrative of specific embodiments of the invention, andvarious uses thereof. They are set forth for explanatory purposes only,and are not to be taken as limiting the invention.

Example 1 Staining Procedure for Biomarkers

Human tumor tissue samples were stained as follows. Tumor tissue in 10%Neutral Buffered Formalin Paraffin blocks are sectioned at 4 microns andthe sections placed onto coated slides. EGFR immunostaining is preformedby using Ventana Medical Instruments, Inc. monoclonal 111.6; HER-2immunostaining is performed by using Ventana Medical Instruments, Inc.monoclonal CB11, and HER-3 immunostaining is performed by using VentanaMedical Instruments, Inc. monoclonal SGP1. Her-1, Her-2, and Her-3 areimmunostained using, for example, the “BenchMark” (VMSI) with I-VIEW(VMSI) detection chemistry. pEGFR immunostaining is performed by usingChemicon monoclonal MB3052. p-ERK (1:100) is obtained from CellSignaling Technology (Beverly, Mass.) and immunostained using a labeledstreptavidin peroxidase technique.

For example, slides for p-ERK (1:100) are antigen retrieved using 0.1 Mcitrate buffer, pH 6.0 in the “decloaker” (Biocare Corp.) and thesections incubated overnight with the primaries at 4° C. The next day,the slides for pERK and pAKT are placed onto the Autostainer (DakoCorp.) and the “LSAB2” kit (Dako) is employed as the detectionchemistry. DAB (Dako) is used as the chromogen. After immunostaining,all immunomarkers, are counterstained manually with 4% ethyl green(Sigma).

Example 2 Procedure for Immunohistochemistry

Quantitative immunohistochemistry (IHC) is performed as previouslydescribed (Bacus, S., et al., Analyt. Quant. Cytol. Histol. 19, 316-328(1997)). EGFR, and erbB-2 immunostaining is performed using thepre-diluted EGFR (Ventana monoclonal 111.6) and erbB-2 (Ventanamonoclonal CB11) antibodies from Ventana on the VMSI automated“BenchMark” staining module as described. The VMSI “I-View” detectionkit is used for all of the VMSI pre-diluted primary antibodies. HER-3 isalso immunostained using the “BenchMark” with I-VIEW detectionchemistry. pErk is immunostained using a labeled streptavidin peroxidasetechnique. Phospho-Erk1/2 slides are antigen retrieved as described(Bacus, S., et al., Analyt. Quant. Cytol. Histol. 19, 316-328 (1997)).Slides are placed onto the Autostainer (Dako Corp.) and the “LSAB2” kit(Dako) employed as the detection chemistry. pEGFR is immunostained in asimilar labeled streptavidin peroxidase technique. pEGFR slides areantigen retrieved with 1 mM EDTA and slides for p-erbB-2 with 0.1Mcitrate buffer, pH 6.0, in the “decloaker”. After staining, EGFR, HER2,HER3, pErk, and pEGFR, are counterstained manually with 4% ethyl green(Sigma). TUNEL assay (Roche Diagnostics, Indianapolis) is performedaccording to the manufacturer's instructions. Investigators preparingand analyzing tissue sections are blinded to both patient tumor type andresponse to therapy.

For IHC, antibodies to EGFR, HER2 and HER3 were from Ventana MedicalScientific Instruments (VMSI) (Tucson, Ariz.); pERK was from CellSignaling Technology Inc. (Beverly, Mass.); anti pEGFR and from Chemicon(Temecula, Calif.).

Example 3 Analysis of Treatment with an Epidermal Growth Factor-DirectedTherapy

53 samples from renal cancer patients enrolled in a clinical trialsponsored by Abgenix and Immunex Corporation for an investigational drugdirected to EGFR were analyzed for expression of various biomarkers. Thesample slides were obtained from Impath Laboratories, Inc.

Immunohistochemical (IHC) analyses were carried out using the automatedstaining devices as described above. The antibodies used for thespecific biomarkers included: Ventana monoclonal 111.6 for EGFR,Chemicon monoclonal MB3052 for pEGFR, polyclonal pERK from CellSignaling Technology for pERK, Ventana monoclonal SGP1 for HER3, andVentana monoclonal CB11 for HER2. For each specimen, a slide was stainedwith control mouse immunoglobulins to establish the existence andlocalization of background staining. In addition, appropriate positivecontrols were run for each IHC stain. Following counterstaining withethyl green, the slides were permanently mounted and analyzed usinginteractive image analysis to establish the optical density ofperoxidase stained cytoplasmic and membrane staining. In the case ofpERK, the fraction of cells expressing nuclear pERK, and the intensityof the stain were measured using a CAS system, and the results wereexpressed as the pERK index (product of OD×percent positive nucleararea). The technician quantifying the results observed areas of tumorthat were not adjacent to normal renal tubules to avoid confounding thequantification. In all cases, the stained slides were viewed by at leasttwo people, including a pathologist and a senior scientist, to establishthat the quantification results were representative of the stainedsections.

Immunohistochemical analyses, quantification, and correlation withresponse data were completed for twenty-nine (29) of the specimens.Partial data, representing analysis of only a subset of the selectedbiomarkers, was available for an additional twelve (12) samples. No datawas obtained on the remaining specimens because of questions as to theidentity of the slides, or the absence of information concerning thepatient's response to ABX-0303. The conclusions that can be drawn fromthe analysis include, but are not limited to, that response to ABX-0303is related to the expression of HER1, and that elevated expression ofHER3 compromises the action of the drug.

Results of the IHC analysis of the renal cancer biopsies, for which atleast HER1 IHC results and clinical response information was available,is presented in Table 1.

TABLE I IHC ANALYSIS HER1 pERK HER3- HER2- HER2 Treatment Pt # HER1“score” pHER1 Index st st cocktail Group Histologic Type Response 3001 8 0 36 17 3 1.0 mg/kg Clear Cell Carcinoma PD 3002 19 +2 20 7 27 5 +2 1.0mg/kg Clear Cell Carcinoma S 3003 8 2 1.0 mg/kg Other MR 3006 6 +1 0 89632 0 +1 Clear Cell Carcinoma PD (focal) 3007 18 +2 6 1127 27 0 0 1.0mg/kg Clear Cell Carcinoma S 3008 19 +2 0 12 15 3 1.0 mg/kg Clear CellCarcinoma PD 3009 0 +2 0 96 15 0 0 1.0 mg/kg Clear Cell Carcinoma PD(focal) 3010 21 +3 0 1100 33 10 PD 3011 5 0 1.0 mg/kg Clear CellCarcinoma S 3012 8 +1 3 40 15 1 0 1.0 mg/kg Clear Cell Carcinoma S 301417 +2 to +3 132 0 1.0 mg/kg Other S 3018 20 +3 9 90 16 0 0 1.0 mg/kgPapillary Carcinoma PD 3019 18 +2 14 924 2 0 1.0 mg/kg Other PR 3020 10+3 3 1176 14 13 1.0 mg/kg Clear Cell Carcinoma S 3031 0 0 5 0 0 0 +1 1.5mg/kg Clear Cell Carcinoma PD 3032 0 +1 1 99 2 0 +1 1.5 mg/kg Clear CellCarcinoma S (very weak) 3033 18 +2 to +3 14 390 50 2 +2 PD 3036 4 +1 0540 0 0 +1 S 3037 8 1.5 mg/kg Clear Cell Carcinoma S 3039 16 +2 0 208 211.5 mg/kg Clear Cell Carcinoma S 3043 15 +2 0 143 40 0 +2 1.5 mg/kgPapillary Carcinoma PD 3047 11 +1 to +2 0 64 37 0 +1 1.5 mg/kg ClearCell Carcinoma S (focal) 3051 18 +2 5 247 1.5 mg/kg Clear Cell CarcinomaS 3053 7 +1 26 0 45 2 1.5 mg/kg Other PD 3065 23 +3 15 108 22 2.0 mg/kgClear Cell Carcinoma S 3068 16 +2 10 221 4 9 +1 2.0 mg/kg Clear CellCarcinoma S 3070 33 +3 0 465 38 15 2.0 mg/kg Clear Cell Carcinoma PD3073 2 0 0 126 23 0 2.0 mg/kg Papillary Carcinoma PD 3075 22 +3 4 77 2813 2.0 mg/kg Clear Cell Carcinoma PD 3077 25 0 2.0 mg/kg Clear CellCarcinoma S 3078 6  0 1 90 11 2.0 mg/kg Clear Cell Carcinoma PD 3080 8+1 21 8 44 11 2.0 mg/kg Clear Cell Carcinoma PD (weak) 3084 20 +3 0 8 345 2.0 mg/kg Clear Cell Carcinoma S 3092 6 +1 5 576 2.5 mg/kg Clear CellCarcinoma S 3095 10 +1 1 0 9 0 +2 MR 3099 13 +2 34 90 13 6 2.5 mg/kgClear Cell Carcinoma S 3101 11 +2 to +3 0 6 25 0 0 2.5 mg/kg Other PD3103 20 +3 0 1134 30 5 +2 2.5 mg/kg Clear Cell Carcinoma PD 3105 15 2.5mg/kg Papillary Carcinoma PD 3108 11 2.5 mg/kg Clear Cell Carcinoma SResults are presented as OD unless otherwise indicated.Based on this analysis, in which the positive and negative predictivevalues were calculated as a function of the optical density, or fractionpositivity, values were determined to stratify samples based uponexpression of the biomarkers analyzed. The results of the analysis ofusing these stratification criteria is presented in Table II.

TABLE II DATA ANALYSIS group of samples (n) RESPONDERS NONRESPONDERS allreported in study (41) 56% 44% HER1 OD > 9 (25) 60% 40% HER1 OD < 10(16) 44% 56% HER1 visual score of +1 or 60% 40% greater (30) pERKindex > 99 (19) 63% 37% pERK index < 100 (16) 38% 62% HER1+/perk− (8)*50% 50% HER1+/perk+ (12) 64% 36% HER3 OD > 9 (26) 38% 62% HER3 OD < 10(7) 86% 14% HER3+/HER1+ (17) 47% 53% HER3+HER1− (9) 22% 78% HER3+/perk+(13) 46% 54% HER3+/perk− (13) 31% 69% HER2 OD > 9 (6) 33% 67% HER2 OD <10 (23) 48% 52% *for purposes of this analysis “+” refers to OD greaterthan 9 upon quantification of HER1, HER2, or HER3; or pERK index ofgreater than 99.Overall, there was no single marker that, when quantified, absolutelycorrelated with response to ABX-0303. This data indicates, however, thatexpression of HER1 and pERK predict response to the drug, while samplesexpressing HER3 are less likely to respond well. The quantitativeanalysis presented assumes that any expression of these markers thatgives an optical density reading of 10 or greater was significant. It isinteresting to note that visual assessment of HER1 staining, where anyintensity of 1+ or greater is considered positive, agrees with thequantification of this marker. Also of interest, only three of thethirty-three samples examined by a pathologist were scored as “0” forHER1 staining intensity, and all three samples were from patients whofailed to respond to ABX-0303. Thus, the absence of detectable HER1(staining intensity “0”), can also be a predictor of response toABX-0303.

The presence of HER3 seems to be a negative predictor of response.Patients whose specimens lacked HER3 by the criterion used here weremore likely to respond than those that had HER3 (86% vs. 38%). There wasno significant correlation between the presence of the phosphorylatedform of HER1 and response to ABX-0303. The lack of pHER1 expression,however, even in samples with significantly elevated levels of HER1, mayhave been a result of a failure to preserve the phosphorylated form ofthis protein during the collection and processing of biopsies. Only 6 ofthe samples analyzed by quantitative IHC were HER2 “positive” by thecriterion of having on OD of 10 or greater. As shown in Table II, thesewere predominantly poor responders to the drug. Interestingly, all sixof these samples had elevated levels of HER3. HER2 expression,quantified using a monoclonal antibody directed against the externaldomain of HER2, was further determined using a cocktail of antibodiesthat recognize both the internal and external domains of the protein.While some additional samples appeared to be positive using thisalternate approach, these observations were not sufficient to confirmthe correlation between HER2 and HER3 expression with lack of responseto ABX-0303.

The expression and co-expression of HER1, HER3, and pERK indicates thatHER1, acting through pERK, was critical to ABX-0303 response, and thatthe action of the drug is compromised in some manner by the presence ofHER3. Notably, biopsies that showed HER3 but not HER1 expression wereless likely to respond to ABX-0303 (22% response rate) than patientswhose tumors expressed both proteins (47% response rate). Dramatically,samples from patients that had low levels of HER3 but expressed HER1and/or pERK at levels of greater than 9, had a 100% response rate toABX-0303. An analysis with a greater number of samples will help confirmany of the conclusions drawn from this analysis. Examples of tumors withhigh and low levels of HER3 are provided in FIGS. 1 and 2.

This data indicates that ABX-0303 effectively targets HER1 to preventthe growth of renal cell cancers. It is not surprising that HER1 seemsto be acting through the MAPK pathway, as shown by the positivecorrelation between pERK expression and response. Of interest is therole of HER3, which was found elevated in 79% of the renal biopsiesanalyzed.

As will be appreciated, the above findings provide useful methods forthe selection of patients for treatment with molecules that target EGFRand predictors of the response of patients. In addition, the abovefindings provide useful methods for the use of ABX-0303. ABX-0303 isdescribed in detail in U.S. Pat. No. 6,235,883 (the disclosure of whichis hereby incorporated by reference) and referred to therein inconnection with the discussions related to hybridoma E7.6.3.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims. All references discussedherein are hereby incorporated by reference in their entirety.

1. A method for predicting a response to an epidermal growth factorreceptor-directed therapy in a human subject, the method comprising thestep of assaying a tumor sample from the human subject before therapywith one or a plurality of reagents that detect expression and/oractivation of predictive biomarkers for cancer; and determining apattern of expression and/or activation of at least two of saidpredictive biomarkers, wherein the pattern predicts the human subject'sresponse to the epidermal growth factor receptor-directed therapy. 2.The method of claim 1, wherein the predictive biomarker is a growthfactor receptor, or a growth factor receptor-related downstreamsignaling molecule.
 3. The method of claim 2, wherein the growth factorreceptor is HER1 (EGFR), pHER1, HER2/neu, HER3, or any combinationthereof.
 4. The method of claim 2, wherein the growth factorreceptor-related downstream signaling molecules is pERK.
 5. The methodof claim 2, wherein the growth factor receptor is HER1 (EGFR), pHER1,HER2/neu, HER3, or any combination thereof, and the growth factorreceptor-related downstream signaling molecules is pERK.
 6. The methodof claim 1, where in the predictive biomarkers are HER1 (EGFR) and HER3.7. The method of claim 6, wherein when HER1 (EGFR) is undetectable ispredictive of the human subject not responding to the epidermal growthfactor receptor-directed therapy.
 8. The method of claim 6, wherein whenHER3 is undetectable is predictive of the human subject responding tothe epidermal growth factor receptor-directed therapy.
 9. The method ofclaim 1, where in the predictive biomarkers are HER1 (EGFR) and pERK.10. The method of claim 1, where in the predictive biomarkers are pERKand HER3.
 11. The method of claim 1, where in the predictive biomarkersare HER1 (EGFR), HER3, and pERK.
 12. A method of selecting a subjectwith cancer for treatment with a molecule targeting epidermal growthfactor receptor (EGFR), comprising determining the level of expressionof HER3 in a cell or tissue sample from the subject, wherein if thelevel of HER3 expression is low in the cells, the subject is selected.13. The method of claim 12, wherein the molecule is an anti-EGFRantibody.
 14. The method of claim 13, wherein the antibody is ABX-0303.15. The method of claim 12, wherein the determining step furthercomprises determining expression of one or more of HER1 (EGFR), pHER1,HER2/neu, and pERK.
 16. A method of predicting the likely response rateto a molecule targeting epidermal growth factor receptor (EGFR) of asubject having a cancer that overexpresses EGFR, comprising the step ofdetermining the level of expression of HER3 in a cell or tissue samplefrom the subject, wherein if the level of HER3 expression is low in thecells, the subject is likely to respond to the molecule targeting EGFR.17. The method of claim 16, wherein the molecule is an anti-EGFRantibody.
 18. The method of claim 17, wherein the antibody is ABX-0303.19. The method of claim 16, wherein the determining step furthercomprises determining expression of one or more of HER1 (EGFR), pHER1,HER2/neu, and pERK.
 20. A method of selecting a subject with cancer fortreatment with a molecule targeting epidermal growth factor receptor(EGFR), the method comprising: a) determining an expression and/oractivation profile of two or more growth factor receptors in cellsand/or tissues of the subject; and b) selecting the subject based on theexpression and/or activation profile, wherein the subject is selectedwhen the level of expression of HER3 is low, the level of expression ofthe HER1 is high, and/or the level of the pERK index is high.
 21. Themethod of claim 20, wherein the molecule is an anti-EGFR antibody. 22.The method of claim 21, wherein the antibody is ABX-0303.
 23. The methodof claim 22, wherein the growth factor receptors comprise one or more ofHER1 (EGFR), pHER1, HER2/neu, and HER3.